Mucopolysaccharidoses and its Diagnosis
Mucopolysaccharidoses (MPS) are a family of inheritable metabolic disorders caused by deficiency of lysosomal enzymes required for degradation of glycosaminoglycans (GAGs). Each known MPS type involves deficiency of a specific lysosomal enzyme required for the stepwise degradation of specific GAGs. GAGs include, for example, chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS).
Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) resulting in accumulation of chondroitin 6-sulfate (C6S) and KS, mainly in cartilage and its extracellular matrix. MPS IVB is caused by deficiency of β-galactosidease, leading to accumulation of KS but not C6S. Clinically, a classic (severe) form of MPS IVA is characterized by systemic skeletal dysplasia such as short trunk dwarfism, kyphoscoliosis, coxa valga, odontoid hypoplasia, abnormal gait, joint mobility problems, restriction of chest wall movement, and a life span of 20-30 years. Patients with an attenuated form can have a nearly normal life span, with mild involvement of the skeleton. See e.g., Dung et al., (2013), Mol Genet Metab 110:129-138. In general, patients with MPS IVB have a milder phenotype of skeletal dysplasia compared with a severe form of MPS IVA.
Mucopolysaccharidosis VII (MPS VII; Sly syndrome) is caused by a deficiency in β-D-glucuronidase. Patients with MPS VII have accumulation of HS, DS, chondroitin-4-sulfate (C4S), and C6S in various tissues and show coarse facial features, mental retardation, short stature, hepatomegaly, bony deformities, GAG excretion, and striking metachromatic granules in peripheral leukocytes (Sly et al. (1973), J Pediatr 82:249-257; Tomatsu et al. (1991), Am J Hum Genet 48:89-96).
Currently, conventional screening methods for MPS are dye-spectrometric methods such as dimethylmethylene blue (DMB) and alcian blue, to measure total GAGs from urine samples. When such urine assays provide positive results, a definitive diagnosis is determined by measuring enzyme activities in white blood cells or fibroblasts. However, current enzyme activity methods cannot be applied to blood and/or tissue extracts without prior protease, nuclease or hyaluroniase treatment. Moreover, total GAGs in urine does not reflect severity of the neurological or skeletal signs and symptoms and substantial overlapping of the total urine GAG level between the age-matched controls and MPS IV patients is observed, resulting in misdiagnosis of the patients.
Methods for measuring specific GAGs in blood by ELISA (Tomatsu et al. (2004), Pediatr Res 55:592-597; Tomatsu et al. (2005), J Inherit Metab Dis 28:187-202) or HPLC (Linhardt et al. (1989), Anal Biochem 181:288-296; Whithman et al. (1999), Glycobiology 9:285-291) have been proposed. These methods are not fit for the mass screening and the cost of performance is expensive. Apart from measuring GAGs, two different approaches have been proposed for MPS detection. One is an immune-capture method for detecting each deficient lysosomal protein from patients with MPS I, MPS II, MPS IIIA and MPS VI (Parkinson-Lawrence et al. (2006), Clin Chem. 52:1660-1668; Tan et al. (2008), Clin Chem 54:1925-1927) and the other is a direct method, assaying individual enzyme activities for MPS I, MPS II, MPS IIIB, MPS IVA, MPS VI and MPS VII patients (Wang et al. (2005), Clin Chem 51:898-900; Gelb et al. (2006), J Inherit Metab Dis 29:397-404; Civallero et al. (2006), Clin Chim Acta 372:98-102; Wang et al. (2007), Clin Chem 53:137-140; Blanchard et al. (2008), Clin Chem 54:2067-2070; Chamoles et al. (2002), Clin Chim Acta 318:133-137; Duffey et al. (2010), Bioorg Med Chem Lett 20:5994-5996; Turecek et al. (2007), Methods Mol Biol 359:143-157; Li et al. (2004), Clin Chem 50:1785-1796). These approaches, which rely on individual antibodies or enzyme activities for first-tier screening, are still being developed to detect all types of MPS, but will be laborious. It may not be feasible to assay simultaneously all corresponding enzyme or protein levels on a large scale. The utility of these methods for screening is limited by the complicated pretreatment steps required prior to performing mass spectrometry, and a different procedure could be required for each metabolite analyzed.
Chondroitin 6-Sulfate (C6S)
CS is involved in specific biological functions including cell adhesion, morphogenesis, neural network formation, and cell division (Sugahara et al. (2003), Curr Opin Struct Biol 13:612-620). Historically, CS was divided into three major subtypes, chondroitin A (chondroitin 4-sulfate; C4S), chondroitin B (dermatan sulfate; DS), and chondroitin C (chondroitin 6-sulfate; C6S), although chondroitin B is not classified as CS any longer. C6S is distributed in the growth plates, especially from the proliferative zone to the hypertrophic zone (Ling et al. (1996) Avian Dis 40:88-98), aorta (Yasuda et al. (2013) Mol Genet Metab 109:301-311), and cornea (Zhang (2005) Invest Ophthalmol Vis Sci 46:1604-1614) in physiological status. C6S has been implicated in pathological status: (1) arterial retention of cholesterol-rich, atherogenic lipoproteins (Mourão et al. (1981) Biochim Biophys Acta 674:178-187), a key event that initiates atherosclerosis (Williams and Tabas (1995) Arterioscler Thromb Vasc Biol 15:551-561); (2) the connective tissue stroma of human colon carcinomas with increase of C6S (Adany (1990) J Biol Chem 265:11389-11396); and (3) urinary excretion of excessive C6S in MPS IVA and VII (Hopwood and Harrison (1982) Anal Biochem 119:120-127; Hata and Nagai (1972) Anal Biochem 45:462-468; (Haskins et al. (1984) Pediatr Res 18:980-984.
To date, CS levels have been determined by HPLC, LC-MS/MS, and capillary electrophoresis, based on the differences in enzymatic digestion using chondroitinase ABC and/or chondroitinase ACII (Imanari et al. (1996), J Chromatogr A 720:275-293; Koshiishi et al. (1998), Anal Biochem 265:49-54; Oguma et al. (2001), Biomed Chromatogr 15:356-362; Karamanous and Hjerpe (2001), Methods Mol Biol 171:181-192; Lamari et al. (2002), Biomed Chromatogr 16:95-102). Although capillary electrophoresis studies showed that a CS spot was visualized subjectively in urine of MPS IVA (Hopwood and Harrison (1982), Anal Biochem 119:120-127; Hata and Nagai (1972), Anal Biochem 45:462-468) C4S and C6S were not separated.
The physiological and pathological roles and distributions of C6S have not been well investigated because of the lack of a rapid, accurate and quantitative method for measurement of that molecule. Thus, no quantitative investigation has been reported on C6S levels in patients with MPS IVA and VII.
There is a need for quantitative molecular marker-based methods for screening individuals for MPS, particularly MPS IVA and MPS VII, and treatment of individuals afflicted with those disorders.